Team:Yale/Our Project/Notebook/Week 7

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lab notebook: week 7 (7/19-7/25)

• Monday 7/19--Investigation of how pre-ligation processing affects ligation results
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Gel Purification of PCR product

• Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold gel extraction protocol to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL
Self-ligation of phsABC to test restriction enzymes

• Took the single enzyme digests from 7/16 and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C
Dual-purpose gel

• Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)





• Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site.
Double digest of gel-purified phsABC

• While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water.
AvaII digestions to check for inhibition

• Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.
• In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above.
Separation of phsABC double digestion

• Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI. Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water.
This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook
• Tuesday 7/20--Self-ligation of single phsABC digestions and serial digestion of ligation components
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Self-ligation of singly digested phsABC fragments

• Each enzyme (AvaII, SpeI, and EcoRI) was used to digest both phsABC that had been purified by gel extraction and phsABC that had been purified by PCR clean-up. Now each of the six resulting solutions is in turn used in a self-ligation reaction containing 17 uL of the digested DNA, 2 uL of T4 ligase buffer, and 1 uL T4 ligase. These ligations are run for four hours at 16˚C to see if a longer ligation time at a lower temperature will yield better results.

Serial digestion of ligation components

• Will try running long sequential digests of both the phsABC insert and the B0015 vector. Will also try omitting CIP. The first B0015 digestion contains of 5 uL of NEB buffer 4, 0.5 uL BSA, 9.7 uL B0015 (at 103.3 ng/uL, total of 1 ug), 1.8 uL XbaI, and 33 uL of water. The first phsABC digestion has 5 uL of NEB buffer 4, 0.5 uL BSA, 7.6 uL of phsABC (1 ug DNA total, derived from PCR purification protocol), 1.8 uL of SpeI, and 35.1 uL of water. Both digestions were run for 4 hours at 37˚C before a twenty minute heat-kill at 80˚C.
• Rather than going through a long purification protocol between steps, simply added 0.5 uL of 5 M NaCl to each digestion so that the salt content fit the parameters needed by EcoRI, then added 1.8 uL of EcoRI and let run another four hours at 37˚C before heatkilling as before.

This day's labwork is also recorded on pages 75 & 76 of the hard copy lab notebook
• Wednesday 7/21--analysis of self-ligation, tranformation of Biobrick Q04121, and set up of ligation attempt #6
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Q04121

• Having settled on Q04121 as an all-in-one promoter system of choice, transform the Biobrick into homemade Xl1-Blue competent cells according to the standard transformation procedure and plate on Kanamycin plates.
Gel of phsABC self-ligation efforts
• Ran a 1.0% agarose gel with the six self-ligation reactions. They were run at 90 V against a 1 kb ladder and pSB74.




From left to right the lanes are: Lane 1--1 kb ladder, Lane 2--circular pSB74 (about 8 kb), Lane 3--ligation of SpeI digest of gel extracted phsABC, Lane 4--Lane 3--ligation of SpeI digest of PCR clean-up phsABC, Lane 5--ligation of AvaII digest of gel extracted phsABC, Lane 6--ligation of AvaII digest of PCR cleanup phsABC, Lane 7--ligation of EcoRI digest of gel extracted phsABC, Lane 8--ligation of EcoRI digest of PCR clean-up phsABC

• The AvaII bands are too faint to see really, but the SpeI and EcoRI ligations appear to show bands near 7.3 kb, where dimers would show if they formed, suggesting that the longer ligation time may have helped. In general though, the gel never resolved well even when the loading dye was run off the end.
Ligation using serial digestion products
• Ethanol precipitated the sequential digest products of B0015 and phsABC from 7/20 according to the standard protocol , then resuspended them each in 30 uL of pH 7.5 TE. Nanodrop gave the resulting concentrations as still very low--12.25 ng/uL for the phsABC and 9.96 ng/uL for the B0015.
• Based on concentrations, devised the following ligation plan for the ethanol precipitated DNA
T4 ligase control ligation --2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.3 uL water
T4 ligase 2:1 insert:vector --2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 5 uL phsABC, 9.3 uL water
T4 ligase 6:1 insert:vector --2 uL ligase buffer, 1 uL T4 ligase, 2.7 uL B0015, 14.8 uL phsABC
Quick ligase control ligation--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL water
Quick ligase 2:1 insert:vector--10 uL ligase buffer, 1 uL Quick ligase, 3.5 uL B0015, 6.5 uL phsABC

• Also did ligations of nonsequentially digested DNA to see if it would work, following the plan outlined below. For the phsABC tried both the gel extraction product and the PCR clean-up product, while with B0015 stuck to the not CIP-treated variety. All reactions were done using normal T4 ligase.
control ligation --2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL water
2:1 gel extraction product --2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 4.7 uL gel extracted phsABC, 9.8 uL water
2:1 PCR cleanup product --2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 5.4 uL PCR clean-up phsABC, 9.1 uL water
6:1 gel extraction product --2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL gel extracted phsABC
6:1 PCR cleanup product --2 uL ligase buffer, 1 uL T4 ligase, 2.5 uL B0015, 14.5 uL PCR clean-up phsABC
Molar ratios are approximate.

• All ligations were allowed to run overnight at 16˚C.
This day's labwork is also recorded on pages 76-79 of the hard copy lab notebook
• Thursday 7/22--Ligation Transformations
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Q02141 transformation

• No growth was observed on the Q04121 plates--may need to find better competent cells.
Transformation of Varied Ligation Efforts
• Retrieved ligations from overnight water bath and heat-killed the T4 ligase with ten minutes at 65˚C.
• After previous ligation troubles, have invested in commercial grade ultracompetent cells. Transformed all of the 7/21 ligations into Top10 One Shot cells according to the standard transformation protocol , also transforming the pUC19 standard that came with the cells. All of these were plated on ampicillin plates, but also transformed DH5alpha cells with Q04121 and plated that with kanamycin.
This day's labwork is also recorded on page 79 of the hard copy lab notebook
• Friday 7/23--Results of ligation transformations
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Ligation Transformants

• The plated Q04121 had two colonies, while the ethanol precipitated DNA ligation control, Quick ligase control, and the ethanol precipitated 2:1 plates all had more colonies than readily countable. The Quick ligase 2:1 had noticeably fewer, and the ethanol precipitated 6:1 ligation gave no colonies at all. In terms of the other transformations, the pUC19, non-ethanol precipitated control, PCR clean-up 6:1 ligation, PCR cleanup 2:1, ligation, and gel extracted 2:1 ligation had lots of colonies, with somewhat fewer on the gel extracted 6:1 ligation plate.
• Chose four colonies from each of the noncontrol ligations and performed colony PCR, lysing the colonies in water before and mixing a uL of the resulting solution with the following: 2 uL VF2 primer (10 mM), 2 uL VR primer (10 mM), 10.8 uL water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the resulting PCR reaction on the "VR" protocol of the thermocycler.
• Also started liquid cultures of each of the colonies in ampicillin LB.
• The colonies were labeled as follows: The four from the Quick Ligase ligation were labeled #1-#4, those from the PCR cleanup 2:1 ligation sere labeled #5-#8, those from the 2:1 ethanol precipitated DNA ligation were called #9-#12, those form the gel extracted 6:1 ligation were #13-#16, while those from the gel extracted 2:1 ligation were numbered #17-#20, and finally those from the PCR clean-up 6:1 ligation were labeled #21-#24

This day's labwork is also recorded on pages 79 & 80 of the hard copy lab notebook
• Saturday 7/24--Gel of colony PCR suggest a successful ligation of phsABC into B0015!
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Gel of Colony PCR

• Ran the twenty-four colony PCR reactions from 7/23 on a single massive 1.0% agarose gel vs a 1 kb ladder at 90 V.

Ladder is very faint in the leftmost lane and the twenty-four reactions are in order from left to right. Despite the faintness, one can see that samples 5, 12, 14, 15, 20, 21 and 24 appear to have the desired 3.6 kb fragment indication a successful ligation.

This day's labwork is also recorded on page 81 of the hard copy lab notebook
• Sunday 7/25--Inoculate cultures of likely ligations successes for miniprep on 7/26
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Inoculation of liquid cultures

• Inoculated liquid ampicillin in LB cultures from the colonies shown to contain successful ligations by the 9/25 gel of colony PCR. Also inoculated a culture of Q04121 for overnight growth, adding kanamycin to a concentration of 50 ng/uL.
This day's labwork is also recorded on page 81 of the hard copy lab notebook